CN1871034B - 用新型腺病毒治疗癌症的方法和组合物 - Google Patents
用新型腺病毒治疗癌症的方法和组合物 Download PDFInfo
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Abstract
本发明是关于一种能高效杀死哺乳动物癌细胞的新型病毒。所述病毒产生一种新型蛋白,该蛋白将两种无毒药物前体转化成有效化疗制剂。所述化疗制剂在局部产生,并帮助所述病毒杀死癌细胞,还使癌细胞对放射线敏感。临床研究已经证明,无论单独使用,还是与药物前体治疗和/或放射治疗联合使用,所述病毒都能有效杀死多种哺乳动物癌细胞。本发明能提供一种安全有效治疗人类癌症的方法。
Description
相关申请的交叉参考
本申请要求于2003年7月9日提交的美国临时专利申请60/486219的优先权。该申请在此一并引入参考。
技术领域
一般地,本发明是关于癌症治疗。更具体地,本发明是关于一种基于腺病毒的癌症治疗。
背景技术
尽管在诊断和治疗方面不断发展,但是每年与癌症相关的死亡数在过去60年中还是没有下降。虽然常规的癌症治疗方法(外科手术、放疗、化疗)在早期疾病患者中得到了很高的治愈率,但是许多癌症复发者和多数晚期癌症患者最终还是死于该疾病。常规癌症治疗的局限不在于它们不能消除肿瘤,而在于它们在消除肿瘤时不能避免对病人的极大伤害。正是这种顾虑限制了外科切除手术的使用范围、放射剂量和照射体积以及化疗药物的剂量和联合使用。如果在肿瘤和正常组织的差异反应方面(即治疗指数)没有显著改善,则提高治疗的效力就没有临床价值。
尽管如此,用于治疗癌症的改进的方法和新制剂已经改善了多种类型癌症患者的存活寿命和存活率。例如,改良的外科手术和放疗方法使得能更加有效地去除局部肿瘤。然而,由于例如肿瘤的部位或转移性癌细胞的扩散,外科手术受到了限制。放疗还受限于其它因素,这些因素限制给药剂量。相对抗辐射的肿瘤在此剂量下不能被治愈。
虽然单一的治疗模式如放疗、化疗、外科手术或免疫疗法能使病人的病情得到改善,但是当这些模式联合使用时,能够达到更好的效果。尤其是能直接作用于含有肿瘤的局部区域的放疗和能提供全身治疗模式的化疗或免疫疗法的联合使用,有利于治疗发生或可能发生疾病扩散的情况。遗憾的是,对于肿瘤细胞相对抗辐射的癌症,放疗的治疗有效性可能有限,因为所述剂量受限于辐射区域正常组织的耐受性。因此需要使癌症肿瘤对放疗的作用敏感化,以便放疗能够更有效地减轻病人体内肿瘤的严重性。此外,开发特异性更强的分离放射部位的治疗方法非常实用,这样可防止放射治疗对健康细胞作用。
相关方法中,腺病毒载体已被用于用所谓“化学基因(chemogene)”对肿瘤细胞进行转导,以减轻某些化学疗法的有害作用。所述化学基因将无毒物质或“药物前体(prodrug)”转化成有毒的治疗有效形式。一些包括基因疗法在内的新方法也正被考虑用于改善癌症治疗的治疗指数。
所述方法之一被称为“自杀基因疗法(suicide gene therapy)”,该方法包括转运和表达非哺乳动物的基因,所述基因编码的酶将无毒药物前体(prodrug)转化成有毒的抗代谢物。目前在临床试验中评价的两种“自杀基因”是大肠杆菌胞嘧啶脱氨酶(E.coli cytosine deaminase,CD)和单纯疱疹病毒1型胸腺嘧啶激酶(herpes simplex virus type-1 thymidine kinase,HSV-1TK)基因,它们分别提供对5-氟胞嘧啶(5-fluorocytosine,5-FC)和9-(1,3-二羟-2-丙氧甲基)鸟嘌呤(ganciclovir,GCV)的敏感性。在所述基因被靶向转运到肿瘤后,所述5-FC和GCV药物前体在局部被转化成有效的化疗制剂,从而导致肿瘤细胞大量死亡(见后文参考文献部分的列表中参考文献1(以及其中引用到的参考文献))。这样就减轻了伴随常规化疗的剂量限制性(dose-limiting)全身毒性。
以前已有将细菌CD和野生型HSV-1 TK基因偶联起来产生新型的CD/HSV-1 TK融合基因(见参考文献部分列表中参考文献1)。所述CD/HSV-1TK融合基因可使CD/5-FC和HSV-1 TK/GCV自杀基因疗法联合应用。早已证明,CD/5-FC和HSV-1 TK/GCV自杀基因疗法可使恶性细胞对特异性药理制剂敏感,并且使恶性细胞对辐射变得明显敏感(见参考文献1-9)。应用含有原型CD/HSV-1 TK融合基因(见参考文献10)的新型消解肿瘤且具有复制能力的腺病毒(Ad5-CD/TKrep),在一些预临床癌症模型(参考文献10-13)以及最近在人前列腺癌病人(参考文献14和15)中,以具有复制能力的腺病毒为媒介的双自杀基因疗法联合和不联合放射疗法的安全性和功效已被证明。
所述以人前列腺癌为靶标的临床试验证明,当与长达三周的5-FC和GCV(vGCV)药物前体治疗联合,同时不联合(参考文献14)和联合(参考文献15)常规剂量(70戈瑞(GY))的三维适形放射治疗(three dimensionalconformal radiation therapy,3DCRT)时,所述Ad5-CD/TKrep病毒的安全剂量高达1012病毒颗粒(Vp)。此外,这些治疗方案已经显示出了临床效果(clinical activity)的体症(参考文献14和15)。
但是尽管有上述进展,仍然十分需要包括用于癌症治疗的有效方法和组合物的发明。根据上述和其它缺点开发了本发明。
发明内容
本发明包括用于治疗癌症的新的、改进的方法和组合物,该组合物包括能有效杀死哺乳动物癌细胞的新病毒。所述病毒产生一种新型蛋白,该蛋白将无毒药物前体转化成有效的化疗制剂。所述化疗制剂在局部产生,并帮助所述病毒杀死癌细胞,还使癌细胞对放射线敏感。临床前研究已经证明,无论单独使用,还是与药物前体治疗和/或放射治疗联合使用,所述病毒都能有效杀死多种人癌细胞。
本发明包括相对以前公开过的原型Ad5-CD/Tkrep病毒而言具有至少两个重大改良的新型“第二代”腺病毒(称为“Ad5-yCD/mutTKSR39rep-ADP”)。Ad5-yCD/mutTKSR39rep-ADP包含改良的yCD/mutTKSR39融合基因,该基因的产物能够更有效地将所述5-FC和GCV药物前体转化成它们的活性化疗制剂。而且,Ad5-yCD/mutTKSR39rep-ADP表达的Ad5 ADP蛋白大大增强具有复制能力腺病毒的肿瘤消解活性。相对于原型Ad5-CD/Tkrep病毒,Ad5-yCD/mutTKSR39rep-ADP在预临床癌症模型中已显示出更强的病毒肿瘤消解和化疗活性。数据显示,本发明的Ad5-yCD/mutTKSR39rep-ADP病毒与5-FC和GCV药物前体治疗以及放射治疗联合使用时,在临床上会表现出低毒性和显著的抗肿瘤活性。
本领域技术人员在仔细研究下列附图和具体描述后,可以明白本发明的其它方面。
附图说明
现通过实施例及其附图描述本发明,其中:
图1为本发明Ad5-yCD/mutTKSR39rep-ADP病毒的示意图;
图2为显示本发明ADP基因的优点的图;
图3A和3B为显示本发明改良的yCD/mutTKSR39基因的优点的图;
图4为显示本发明ADP基因的优点的图;
图5显示了Ad5-yCD/mutTKSR39rep-ADP在前列腺内LNCap C4-2小鼠模型中的Kaplan-Meier统计图。
具体实施方式
一般地,本发明包括用于治疗癌症的方法和组合物。更具体地,本发明提供了一种在给予药物前体时能杀死癌细胞并使残存癌细胞对放射线更敏感的治疗方法。
本发明的实施方式包括一种新型病毒,该病毒产生将无毒药物前体转化成化疗制剂的蛋白。所述药物前体可以在治疗时局部产生或给予。优选情况下,所述病毒为消解肿瘤且具有复制能力的腺病毒,例如但不限于Ad5-yCD/mutTKSR39rep-ADP。当需要此类治疗的病人被给予腺病毒时,该腺病毒将至少两种药物前体转化成化疗制剂。所述药物前体可以包括但不仅限于5-氟胞嘧啶和9-(1,3-二羟-2-丙氧甲基)鸟嘌呤(ganciclovir,GCV及其衍生物)。
除将药物前体转化成化疗制剂的能力之外,本发明的实施方式还可以使癌细胞对放射线敏感。通过使癌细胞敏感化,可以使用更低剂量的放射线而不限制放射线的优点。此外,由于癌细胞对放射线更为敏感,放疗更为有效;同时正常细胞并不敏感,因此限制了癌症治疗的副作用。本发明的治疗方法可以与其它治疗方法联合使用,所述其它治疗方法如外科手术、化疗、激素治疗和免疫疗法。
在优选的实施方式中,本发明包括一种新型消解肿瘤且具有复制能力的腺病毒(Ad5-yCD/mutTKSR39rep-ADP),该病毒包括酵母胞嘧啶脱氨酶(yeastcytosine deaminase,yCD)/突变体SR39单纯疱疹病毒1型胸腺嘧啶激酶(mutant SR39 herpes simplex virus type-1 thymidine kinase,mutTKSR39)融合基因和腺病毒5型(adenovirus type 5,Ad5)的腺病毒死亡蛋白(adenovirusdeath protein,ADP)基因。Ad5-yCD/mutTKSR39rep-ADP在人癌细胞中复制,并高效杀死人癌细胞。Ad5-yCD/mutTKSR39rep-ADP产生新型yCD/mutTKSR39融合蛋白,该蛋白将两种药物前体5-氟胞嘧啶(5-fluorocytosine,5-FC)和9-(1,3-二羟-2-丙氧甲基)鸟嘌呤(ganciclovir,GCV;以及GCV衍生物)转化成有效的化疗制剂(指双自杀基因疗法)。yCD/5-FC和HSV-1 TKSR39自杀基因疗法都显示出了有效的化疗活性并都能使肿瘤细胞对电离辐射敏感。
仅仅通过实施例,临床前研究表明所述Ad5-yCD/mutTKSR39rep-ADP病毒在自身单独使用,或者与双自杀基因疗法和/或放射疗法联合使用时都能有效杀死多种人癌细胞。在临床情况下,所述Ad5-yCD/mutTKSR39rep-ADP病毒可凭借其病毒介导肿瘤消解作用而用作单一治疗手段,也可以与yCD/5-FC和HSV-1 Ad5-TKSR39/GCV自杀基因疗法联用,起到病毒消解肿瘤/化疗联合作用,或与yCD/5-FC和HSV-1 Ad5-TKSR39/GCV自杀基因疗法以及放射疗法联用,起到病毒消解肿瘤/化疗/放射敏化联合作用(指三联疗法)。三联疗法能结合人癌处理中的其它常规癌症治疗方法例如:外科手术、化疗、激素疗法和免疫疗法。
为进一步开发这种基于基因疗法的途径作为治疗癌症的方法,一种新型“第二代”腺病毒(“Ad5-yCD/mutTKSR39rep-ADP”)已被开发出来,该新病毒相对于原型Ad5-CD/Tkrep病毒而言,具有两个重大的改良。Ad5-yCD/mutTKSR39rep-ADP包含改良的yCD/mutTKSR39融合基因,该基因的产物能够更有效地将所述5-FC和GCV药物前体转化成它们的活性化疗制剂。而且,Ad5-yCD/mutTKSR39rep-ADP表达的Ad5 ADP蛋白大大增强具有复制能力腺病毒的肿瘤消解活性。相对于原型Ad5-CD/Tkrep病毒,Ad5-yCD/mutTKSR39rep-ADP在预临床模型中已显示出更强的病毒引起的肿瘤消解和化疗活性。
与列出的其它方法相比,本发明的通过病毒转染引入核苷酸的方法具有几个优点。由病毒感染特性可以获得更高的有效率。此外,病毒高度特化,并且一般在特定细胞类型中感染和增殖。因此,在活体内或在组织或细胞的混合培养物中,它们的天然特异性可被用于使载体靶向特定的细胞类型。病毒载体还可被特定受体或配体修饰,从而通过受体介导事件改变靶向特异性。
再者,附加的特征也可以被加入到载体中,以确保载体的安全性和/或增强载体的治疗效果。这些特征包括例如能用于对感染有重组病毒的细胞进行阴性选择的标记。此类阴性选择(negative selection)标记的实例之一为上述TK基因,该基因提供对抗生素9-(1,3-二羟-2-丙氧甲基)鸟嘌呤(ganciclovir)的敏感性。因此阴性选择为一种方法,通过该方法使感染得到控制,因为该方法通过加入抗生素而提供可诱导的自杀。这样的保护确保,例如若发生可改变病毒载体或重组序列的变异,将不会发生细胞的转化。
限制对特定细胞类型的表达的特征,也包括在某些实施方式中。例如所述特征包括目标细胞类型的特异的启动子和调控元件。
此外,重组病毒载体在本发明核苷酸体内表达中很有用,因为所述载体具有例如横向感染和靶向特异性的优点。横向感染为诸如逆转录病毒的生活史所固有的,它通过单个被感染细胞产生很多子代病毒体,该子代病毒体出芽逸出并感染邻近的细胞。结果导致大面积迅速感染,大多数被的细胞最初不是被原始病毒粒子感染。这与垂直型感染相反,所述垂直型感染中致病因子(Infectious agent)仅通过子代传播。不能横向传播的病毒载体也能产生。若预期目标是仅在局部某些靶标细胞中导入特定基因,则上述特点非常有用。
如上所述,病毒是特异性很强的致病因子,大多数情况下,病毒已进化以躲避宿主防御机制。病毒一般在特定细胞类型中感染和增殖。病毒载体的靶向特异性利用其对特异性靶标预定细胞类型的天然特异性,从而可将重组基因导入被感染细胞中。用于本发明方法中的载体取决于靶向目标细胞类型,并为本领域人员公知。例如,若乳癌为靶标,则应用对这种上皮细胞有特异性的载体。同样地,若造血系统的疾病或病理状态为靶标,则应用对血细胞和它们的前身,优选对造血细胞特异类型,有特异性的病毒载体。
重组载体能以几种方式给药。例如,所述给药途径能利用病毒载体的靶向特异性,从而无须在发病部位进行局部给药。但是局部给药能提供更快更有效的治疗。给药也可以通过例如静脉或皮下注射到受治疗者体内进行。注射之后,病毒载体将在体内循环,直到它们识别适合于感染的靶向特异性宿主细胞。
可替换的给药方式可以是直接局部接种到疾病或病理条件的部位,或接种到给上述部位供应营养的脉管系统中。局部给药是有利的,因为没有稀释效应,因此只需较小剂量就可以在多数靶标细胞中得到表达。局部接种还能减少其它给药形式所要求的靶向必要条件,因为能采用可感染接种区域内所有细胞的载体。若只期望在接种区域内特定亚型的细胞中表达,则目标细胞亚型的特异启动子和调控元件可以用来达到此目标。例如,所述非靶向载体可以是病毒载体、病毒基因组、质粒、噬菌粒等。转染媒介物如脂质体也可以用于将上述非病毒载体导入到接种区域内的受体细胞中。所述转染媒介物为本领域技术人员公知。
考虑到病人个体的临床条件、给药的方式和部位、给药时间安排、病人年龄、性别、体重和其它执业医生公知的因素,本发明的化合物依照《优质医疗指南》(Good Medical Practice)确定给药方式和给药剂量。在这里为了达到治疗目的的药物“有效量”因此取决于本领域公知的考虑事项。所述量必需有效改善病情,所述改善包括但不限于改善存活率或更快的恢复、或改善或消除症状和本领域技术人员选择适于测量的其它指标。
本发明提供的方法中,本发明的化合物能以多种方式给药。应该注意的是本发明的化合物可以作为化合物给药,并且可以单独给药或作为活性成分与药物适合载体、稀释剂、辅药和赋形剂结合给药。所述化合物可以口服给药、皮下或非肠道给药包括静脉内给药、动脉内给药、肌内给药、腹膜内给药和鼻内给药以及鞘内给药和输液技术。植入所述化合物也有作用。接受治疗的对象是温血动物,特别地,包括人类在内的哺乳动物。药物适合载体、稀释剂、辅药和赋形剂以及植入载体一般指不与本发明所述活性成分反应的惰性无毒固体或液体填料、稀释剂或形成胶囊的物质。
值得注意的是,治疗人类的疗程通常比在此用作示例的小鼠或其它实验动物长,治疗时间长度与疾病进程的时间长短和药物效力成比例。所述剂量可以是隔几天时间的单一剂量或复合剂量。治疗时间长度一般与疾病进程的时间长短和药物效力以及接受治疗的病人类型成比例。
当本发明的化合物非肠道给药时,该化合物一般制成单位剂量的注射剂型(溶液剂型、悬液剂型、乳剂剂型)。适合注射的药物剂型包括无菌水溶液或分散体以及重制无菌注射液或分散体用的无菌粉末。所述载体可以是溶剂或分散介质,例如包括水、乙醇、多羟基化合物(例如:丙三醇、丙二醇、液态聚乙二醇等)、它们适当的混合物以及植物油。
为能保持适当的流动性,例如可以使用诸如卵磷脂包埋、在分散体中保持必要的粒度以及使用表面活性剂。非水赋形剂如棉子油、芝麻油、橄榄油、豆油、玉米油、葵花油或花生油以及酯例如异丙基肉豆蔻酸酯,也可以用作化合物组合物的溶剂系统。另外,能加入多种增强组合物稳定性、无菌性和等渗性的添加剂,包括抗菌防腐剂、抗氧化剂、熬合剂以及缓冲液。通过多种抗细菌和抗真菌制剂,例如对羟基苯甲酸酯(parabens)、氯代丁醇、苯酚和山梨酸等可确保防止微生物作用。多数情况下适当包括等张剂,例如糖类、氯化钠等。使用延迟吸收剂如一硬脂酸铝和明胶,能得到注射药物剂型的长效吸收。然而根据本发明,所有使用的赋形剂、稀释剂或添加剂必须与所述化合物相适合。
无菌注射溶液能通过将本发明所利用的化合物,混合到所需量的含有多种其它所需成分的适当溶剂中制备。
本发明的药理制剂能以注射剂型对病人给药,该注射剂型含有任何适合的载体例如各种赋形剂、辅药、添加剂和稀释剂;或者本发明所利用的化合物能非肠道对病人给药,所述非肠道给药方式可以是皮下缓释植入体或靶向给药系统如单克隆抗体、载体给药(vectored delivery)、离子渗透、聚合体基质、脂质体和微球体。许多其它此类植入体、给药系统和模式(modules)为本领域技术人员公知。
在一种实施方式中,本发明的化合物能最初通过静脉内注射使药物在血液中达到适当的水平。然后通过口服剂型维持病人药物水平,但是可以根据病人情况和上述说明采用其它给药形式。给药量因接受治疗的病人不同而不同。
除非上下文特别说明或暗示,下列术语和短语具有下面提供的含义。
本文中所用术语“基因疗法(gene therapy)”指将感兴趣的遗传物质(genetic material of interest)(如DNA或RNA)转移到宿主内,以治疗或预防遗传性或获得性疾病或条件表现型。所述感兴趣的遗传物质编码的产物(例如:蛋白质、多肽、肽、功能RNA、反义RNA)为有机体所需要。例如所述感兴趣的遗传物质能编码有治疗价值的激素、受体、酶、多肽或肽。所述感兴趣的遗传物质还可以编码自杀基因。大体上参考《基因疗法(genetherapy)》正文(Advances in Pharmacology 40,Academic Press,1997)。
短语“体内基因疗法(in vivo gene therapy)”指被转移的遗传物质被原位引入到受治有机体的靶细胞中,所述原位指在受治体内。治疗后,遗传上发生改变的靶细胞原位表达被转染的遗传物质。如果宿主基因有缺陷,这种治疗也包括原位修复缺陷基因。
短语“基因表达媒介物(gene expression vehicle)”指任何能将异源核酸递呈/转移到宿主细胞内的媒介物。所述表达媒介物可以包括本领域公知的以细胞选择方式控制靶向、核酸的表达和转录的元件。应当注意的是,基因的5’非翻译区(untranslated region,5’UTR)和/或3’非翻译区(3’UTR)通常可以被表达媒介物的5’非翻译区和/或3’非翻译区所替代。因此,这里用到的表达媒介物按照要求不包括被转移实际基因的5’非翻译区和/或3’非翻译区,只包括特定氨基酸编码区域。表达媒介物可以包括控制异源物质转录的启动子,以及允许选择性转录的组成型或诱导型启动子。可以选择性地包括达到必要转录水平所必需的增强子。增强子一般为非翻译DNA序列,该序列持续与编码基因(顺势,in cis)起作用,改变受所述启动子控制的基础转录水平。所述表达媒介物还可以包括选择基因。
实施例1 Ad5-yCD/mutTKSR39rep-ADP腺病毒的描述
Ad5-yCD/mutTKSR39rep-ADP腺病毒全部和部分的DNA和翻译的蛋白序列、yCD/mutTKSR39融合基因和ADP基因(SEQ ID NO.1至SEQ ID NO.5)已在下面的参考文献区域列表中公开。鉴于上述序列提出下述实施例。
实施例中所述Ad5-yCD/mutTKSR39rep-ADP腺病毒(SEQ ID NO.1)是具有复制能力的5型腺病毒(对本领域技术人员而言,该病毒的序列是已经公知的并且是可以得到的),该病毒包括E1区域中改良的yCD/mutTKSR39融合基因以及E3区域中的Ad5 ADP基因。Ad5-yCD/mutTKSR39rep-ADP的图示在图1中提供(图1中,“CMV”=人类巨细胞病毒启动子;“SV40”=猿猴病毒40聚腺苷酸化序列;和“mu”=图谱单位(map units))。正如图1所示,CMV-yCD/mutTKSR39-SV40表达盒(expression cassette)位于E1区域,代替了缺失的55千道尔顿(kDa)的EIB基因。CMV-ADP-SV40表达盒位于E3区域,代替了缺失的E3基因。
Ad5-yCD/mutTKSR39rep-ADP在55千道尔顿(kDa)的E1B基因中存在1255个碱基对(bp)的缺失(碱基2271至3524)(见SEQ ID NO.2)。使用本领域普通技术人员公知的方法,将两个超前翻译终止密码子(prematuretranslation stop codon)设计在55千道尔顿(kDa)的E1B基因中,导致产生缩短的78个氨基酸的无功能E1B蛋白。Ad5-yCD/mutTKSR39rep-ADP表达野生型的Ad5 E1A和19千道尔顿(kDa)的E1B蛋白。所述yCD/mutTKSR39融合基因(SEQ ID NO.4)被插入取代被缺失的55千道尔顿(kDa)的E1B基因。所述yCD/mutTKSR39融合基因的表达通过人类巨细胞病毒(CMV)启动子驱动,并且利用猿猴病毒40(SV40)聚腺苷酸化元件。所述yCD/mutTKSR39融合基因编码59千道尔顿(kDa)的yCD/mutTKSR39融合蛋白,该蛋白能作为酶催化将5-氟胞嘧啶(5-fluorocytosine,5-FC)转化成为氟尿嘧啶(fluorouracil,5-FU)并且将9-(1,3-二羟-2-丙氧甲基)鸟嘌呤(ganciclovir,GCV)及其衍生物转化成它们相应的一磷酸盐(如GCV-MP)。5-FU和GCV-MP的下游代谢产物为DNA复制的有利抑制剂,可导致正在分裂的细胞死亡。这些下游代谢产物还是有利的辐射敏化剂,能显著增强放射治疗的治疗效果(见参考文献1至14)。表达yCD/mutTKSR39融合蛋白的细胞以及通过旁观者效应(bystander effect)的邻近细胞,可被yCD/5-FC和HSV-1 TKSR39/GCV自杀基因疗法杀死,并且敏感于电离辐射的杀伤作用。
Ad5-yCD/mutTKSR39rep-ADP还包括E3区域内的一段2.8千碱基对(kb)的缺失(碱基28133到30181),被缺失的部分影响抑制宿主免疫应答的基因,但不是病毒复制所必需的(见SEQ ID NO.3)。Ad5-yCD/mutTKSR39rep-ADP包括Ad5 ADP表达盒,代替天然的Ad5 E3基因。ADP基因(SEQ ID NO.5)的表达通过人类巨细胞病毒(CMV)启动子驱动,并且利用猿猴病毒40(SV40)聚腺苷酸化元件。真正的111.6千道尔顿(kDa)的Ad5 ADP蛋白产生出来,该蛋白可显著增强具有复制能力的腺病毒的肿瘤消解活性。Ad5-yCD/mutTKSR39rep-ADP缺乏其它公知的Ad5 E3基因(gp19、10.4kDa、14.5kDa和14.7kDa的基因)。
实施例2 Ad5-yCD/mutTKSR39rep-ADP腺病毒的构建
用于构建Ad5-yCD/mutTKSR39rep-ADP的含有腺病毒序列的质粒得自Microbix公司(多伦多,加拿大)。为产生pCMV-yCD/mutTKSR39表达质粒(左端载体,left-end vector),使用线性化的pET23d:HSVTKSR39作为模板,通过聚合酶链式反应(polymerase chain reaction,PCR)产生突变体SR39单纯疱疹病毒1型胸腺嘧啶激酶基因(HSV-1 TK gene)(参考文献16)。下列引物对(primer pair)用于产生mutTKSR39的PCR产物:
5′-GATCGGATCCCTCGAGATC2CTAGCATGGCTTCGTACCCCGGC-3
5′-GATCGAATTCTTCCGTGTTTTCAGTTAGCCTC-3
将得到的1128碱基对(bp)片段用BamHI(GGATCC)+EcoRI(GAATTC)消化,在pCA14-CDglyTK-E1aE1b(参考文献10)的BamHI+EcoRI位点间克隆,产生pCA14-CMV-mutTKSR39-ElaElb,所述pCA14-CDglyTK-ElaElb已去除原型CD/HSV-1 TK融合基因。使用线性化的pBAD-ByCD作为模板,通过聚合酶链式反应(polymerase chain reaction,PCR)产生酵母胞嘧啶脱氨酶基因(yCD gene)(参考文献17)。下列引物对(primer pair)用于产生yCD的PCR产物:
5′-GATCCTCGAGCCACCATGGTGACAGGGGGAATG-3′
5′-GATCGCTAGCACCTCCCCCACCGCCTCtCCCTCCACCCTCACCAATATCTTC-3’
将得到的526碱基对(bp)片段用XhoI(CTCGAG)+NheI(GCTAGC)消化,并在pCA14-CMV-mutTKSR39-ElaElb的XhoI+NheI位点间克隆,产生pCA14-CMV-yCD/mutTKSR39-ElaElb。
为产生pBHG10-Paclmod-CMV-ADP(右端载体,right-end vector),通过聚合酶链式反应(polymerase chain reaction,PCR)产生ADP基因,并将该基因克隆到pBHG10-PacImod的PacI和SwaI位点间。pBHG10-PacImod为pBHG10的衍生物(Microbix;Toronto,Canada)并且在E3区域内包括PacI和SwaI位点以便定向克隆。
pBHG10是一种质粒,该质粒包括不含E1区域内碱基188至1339和E3区域内碱基28133至30818的整个腺病毒5型基因组。用野生型Ad5 DNA作为模板,得到含有ADP基因的333个碱基对(bp)的PCR产物。下列引物对(primer pair)用于产生ADP的PCR产物:
5′-GATCGGATCCCCTGCTCCAGAGATGACCGGC.3’
5′-GATCAAGCTTGGAATCATGTCTCAMAATC-3′
将得到的333碱基对(bp)PCR产物用BamHI(GGATCC)+HindIII(AAGCTT)消化,克隆到BamHI-HindIII消化的pCA14(Microbix;Toronto,Canada)中,产生pCA14-ADP。下列引物对(primer pair)用于通过PCR产生整个CMV-ADP-SV40多聚腺苷酸表达盒(polyA expression cassette):
5′-GATCATTTAAATAATTCCCTGGCATTATGCCCAGTA-3′
5′-GATCTTAATTAATCGATGCTAGACGATCCAGACATG-3′
在5′引物的CMV启动子上游引入SwaI限制性位点(ATTTAAAT),并在3′引物的SV40 poly A区域下游引入PacI限制性位点(TTAATTAA)。所得PCR产物用SwaI和PacI消化,克隆到SwaI-PacI消化的pBGH10-PacImod中,产生pBGH10-PacImod-CMV-ADP。
为产生Ad5-yCD/mutTKSR39rep-ADP病毒,将pCA14-CMV-yCD/mutTKSR39-ElaElb(10微克)通过PvuI消化线性化,并使用CaPO4-DNA沉淀方法使之与ClaI线性化的pBHG10-PacImod-CMV-ADP(30微克)共转染HEK293细胞(人胚肾细胞)(Microbix)。分离的空斑在7-14天后收获,并第二次在HEK293细胞中空斑纯化。病毒型二次纯化空斑用于转染HEK293细胞产生病毒粗悬液和CsCl梯度纯化腺病毒。
实施例3 Ad5-yCD/mutTKSR39rep-ADP包括的ADP基因在体外的优势
人类DU145前列腺腺癌(prostate adenocarcinoma)细胞以5×104个细胞/孔的浓度在24孔板上培养,并感染分级量的Ad5-CD/TKrep(泳道1)和Ad5-yCD/mutTKSR39rep-ADP(泳道2)。5天后,固定细胞并用结晶紫染色。结果(如图2所示,“Vp”=病毒粒子)清楚地表明,包括Ad5 ADP基因并表达ADP蛋白的具有复制能力的腺病毒(即Ad5-yCD/mutTKSR39rep-ADP)与缺乏ADP的腺病毒相比具有相当强的肿瘤消解活性。换句话说,Ad5 ADP基因的存在显著增强了具有复制能力的腺病毒的肿瘤消解活性。上述结果显示了Ad5-yCD/mutTKSR39rep-ADP包括的ADP基因在体外的优势。
实施例4 Ad5-yCD/mutTKSR39rep-ADP包括的yCD/mutTKSR39基因在体外的优势
A.CD的分析
将前列腺癌细胞(LNCaP C4-2)假感染(mock-infected)(泳道1和泳道5)或以10感染复数(Multiplicity OfInfection,MOI)感染Ad5-CD/TKrep(泳道2和泳道6)、Ad5-yCD/mutTKSR39rep-ADP(泳道3和泳道7)、Ad5-yCD/mutTKSR39rep-hNIS(泳道4和泳道8)。七十二小时后,通过使用[14C]-胞嘧啶(泳道1-4)和[3H]-5-FC(泳道4-8)作为底物,检测细胞的CD活性。结果在图3A中表示[胞嘧啶(较低左箭头)、尿嘧啶(较高左箭头)、5-FC(较高右箭头)、5-FU(较低右箭头)]。正如图3A所示,表达改良yCD/mutTKSR39基因的重组腺病毒,例如Ad5-yCD/mutTKSR39rep-ADP,与表达包含在原型Ad5-CD/TKrep病毒中的CD/HSV-1 TK融合基因的腺病毒相比,前者显示出5-FC到5-FU更大量的转化,但是并不将胞嘧啶转化为尿嘧啶。
B.细胞病理效应分析
将细胞(106个细胞,60毫米培养皿)假感染(mock-infected)或以3感染复数感染Ad5-CD/TKrep、Ad5-yCD/mutTKSR39rep-ADP。次日,细胞重新在培养基中平板培养(24孔板),所述培养基含有多种微克/毫升级浓度的5-FC(孔3-7和15-19,从左到右,从上到下计)或GCV(孔8-12和20-24,从左到右,从上到下计)。9天后细胞用结晶紫染色。结果(如图3B所示)表明表达改良yCD/mutTKSR39基因的重组腺病毒,例如Ad5-yCD/mutTKSR39rep-ADP,与表达包含在原型Ad5-CD/TKrep病毒中的CD/HSV-1 TK融合基因的腺病毒相比,当与5-FC药物前体结合时,前者使更大量的细胞死亡。图3A和图3B所示的结果综合在一起显示出Ad5-yCD/mutTKSR39rep-ADP包括的yCD/mutTKSR39基因在体外的优势。
本实施例的结果还表明yCD/5-FC和HSV-1 TKSR39/GCV自杀基因疗法可以用于增强Ad5-yCD/mutTKSR39rep-ADP病毒自身的疗效。Ad5-yCD/mutTKSR39rep-ADP包括一种新型yCD/mutTKSR39融合基因,该基因的产物相对于原型Ad5-CD/Tkrep病毒产生的CD/HSV-1 TK融合蛋白而言,具有改良的催化活性。与表达原型CD/HSV-1-TK融合蛋白的腺病毒相比,表达改良yCD/mutTKSR39融合蛋白的重组腺病毒显示出5-FC到5-FU更大量的转化,并可能更多的将GCV转化为GCV-MP。因此,yCD/5-FC和HSV-1TKSR39/GCV自杀基因疗法可以单独应用,并且联合使用能增强Ad5-yCD/mutTKSR39rep-ADP病毒的肿瘤破坏作用。
实施例5 Ad5-yCD/mutTKSR39rep-ADP包括的ADP基因在体内的优势
在第0、2和4天(图4中箭头)对肌肉(腿部)C33A肿瘤(150-200立方毫米)注射1010病毒粒子(vp)的Ad5-CD/TKrep或Ad5-CD/TKrep-ADP。在第5-11天(图4中阴影条)给予5-FC(500毫克/千克/天)和GCV(30毫克/千克/天)。每隔一天检测肿瘤体积。预定的终点是500立方厘米。存活定义为90天后动物没有肿瘤(治愈)或肿瘤小于500立方毫米。结果(如图4和下表1所示)显示对体内肿瘤细胞更强的破坏作用,因而表明了Ad5-yCD/mutTKSR39rep-ADP包括的ADP基因在体内的优势。换句话说,Ad5ADP基因的存在显著增强了具有复制能力的腺病毒在体内和体外的肿瘤消解活性。
a中值存活期以天计算。
bAd5-CD/TKrep-ADP对Ad5-CD/TKrep
cAd5-CD/TKrep-ADP+5-FC+GCV对Ad5-CD/TKrep+5-FC+GCV
实施例6 Ad5-yCD/mutTKSR39rep-ADP在小鼠模型体内的效果
患有前列腺内LNCaP C4-2肿瘤(肿瘤大小25-50立方毫米)的雄性重症联合免疫缺陷小鼠(SCID)在第0天注射约109病毒粒子(vp)的Ad5-yCD/mutTKSR39rep-ADP(图5中箭头)。在第3-9天给予5-FC(500毫克/千克/天)和GCV(30毫克/千克/天)(图5中阴影条)。每周测量前列腺特异抗原(PSA)血清。预定的终点是PSA=500纳克/毫升(ng/ml)。结果(如图5和下表2所示)显示,使用本发明的Ad5-yCD/mutTKSR39rep-ADP增加小鼠模型中值存活期的时间和/或肿瘤治愈。
aAd5-yCD/mutTKSR39rep-ADP对PBS;
bAd5-yCD/mutTKSR39rep-ADP+5-FC+GCV对PBS
实施例7用yCD/5-FC和HSV-1 TKSR39/GCV使人癌细胞放射敏感化
正如发明人先前的实验所示(见参考文献1-14),yCD/5-FC和HSV-1TKSR39/GCV自杀基因疗法也可以用于使人癌细胞放射敏感化。Ad5-yCD/mutTKSR39rep-ADP包括新型yCD/mutTKSR39融合基因,相对于原型Ad5-CD/Tkrep病毒产生的CD/HSV-1 TK融合蛋白而言,该新型yCD/mutTKSR39融合基因的产物具有改良的催化活性。已有的研究表明CD/5-FC和HSV-1 TK/GCV自杀基因疗法能使人肿瘤细胞对辐射电离敏感化。因此,由于Ad5-yCD/mutTKSR39rep-ADP表达改良的yCD/mutTKSR39融合蛋白,因此可以导致肿瘤细胞在体内的放射敏感性更强。
贯穿本申请,不同参考文献标注以不同的文献标号。这些文献标号列表及其完整的引用出处在后文给出。这些参考文献全文的公开因此也作为参考并入本申请,以更充分地描述本发明所属技术领域的发展状态。
尽管本发明已通过前述优选和可选实施方式和实施例得以详细展示和描述,但本领域技术人员应该理解的是,在此描述的本发明的实施方式的各种替代方式也可以实现本发明,且不偏离本发明权利要求书定义的精髓和范围。这意味着权利要求书定义的本发明范围以及所述权利要求书范围内的方法和组合物,以及与它们等同的方法和组合物都在权利要求书保护范围内。应该理解本发明的说明书包含这里所述的所有新型和非显而易见的元素的组合,该申请以及随后的申请的权利要求将包括这些元素的任何新型和非显而易见的组合。前述实施方式是例证性的,没有任何单一特征或元素对该申请或随后申请要求保护的所有可能组合是必要的。权利要求中陈述了“一种”或“一种首要的”元素及其等同物,这种权利要求应该理解包括一种或多种此类要素在内,既不必需也不排除两个或两个以上此类要素。
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Claims (9)
1.一种分离的多核苷酸,该多核苷酸包括如SEQ ID NO:4所示的酵母胞嘧啶脱氨酶/突变体SR39单纯疱疹病毒1型胸腺嘧啶激酶融合基因的核苷酸序列,该多核苷酸还包括如SEQ ID NO:5所示的腺病毒5型腺病毒死亡蛋白基因。
2.一种分离的多肽,该多肽包括由权利要求1所述的多核苷酸编码的氨基酸序列,该多肽将药物前体5-氟胞嘧啶和9-(1,3-二羟-2-丙氧甲基)鸟嘌呤转化成活性化疗制剂。
3.根据权利要求2所述的多肽,其中,所述多肽包括SEQ ID NO.4所示的氨基酸序列。
4.一种重组腺病毒,该病毒包括权利要求1所述的多核苷酸。
5.根据权利要求4所述的重组腺病毒,其中,所述腺病毒为具有复制能力的5型腺病毒。
6.根据权利要求4所述的重组腺病毒,其中,所述重组腺病毒包括SEQID NO.1所示的核苷酸序列。
7.一种药物组合物,该组合物包括权利要求4-6中的任意一项所述的重组腺病毒和药物适合载体。
8.权利要求4所述的重组腺病毒在制备用于治疗哺乳动物的恶性肿瘤的药物组合物中的应用。
9.一种将5-氟胞嘧啶和/或9-(1,3-二羟-2-丙氧甲基)鸟嘌呤转化成活性化疗制剂的方法,该方法包括将权利要求2所述的多肽与5-氟胞嘧啶和/或9-(1,3-二羟-2-丙氧甲基)鸟嘌呤接触。
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- 2004-07-09 KR KR1020067000479A patent/KR100903729B1/ko active IP Right Grant
- 2004-07-09 CN CN2004800243239A patent/CN1871034B/zh not_active Expired - Lifetime
- 2004-07-09 US US10/888,492 patent/US20090285783A1/en not_active Abandoned
- 2004-07-09 JP JP2006518967A patent/JP5550803B2/ja not_active Expired - Lifetime
Patent Citations (2)
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WO2001004282A2 (en) * | 1999-07-12 | 2001-01-18 | Saint Louis University | Replication-competent anti-cancer vectors |
CN1379109A (zh) * | 2001-10-29 | 2002-11-13 | 上海三维生物技术有限公司 | 特异杀伤原发肝癌细胞的腺病毒载体及使用方法 |
Non-Patent Citations (4)
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FREYTAG , ET AL,.A NOVEL THREE-PRONGED APPROACH TO KILLCANCER CELLS SELECTIVELY: CONCOMITANT VIRAL,DOUBLE SUICIDE GENE, AND RADIOTHERAPY.HUMAN GENE THERAPY10.1998,101323-1333. |
FREYTAG, ET AL,.A NOVEL THREE-PRONGED APPROACH TO KILLCANCER CELLS SELECTIVELY: CONCOMITANT VIRAL,DOUBLE SUICIDE GENE, AND RADIOTHERAPY.HUMAN GENE THERAPY10.1998,101323-1333. * |
KENNETH N, ET AL,.GENIS:GENE EXPRESSION OF SODIUM LODIDESYMPORTER FOR NONINVASIVE IMAGING OF GENETHERAPY VECTORS AND QUANTIFICATION OF GENEEXPRESSION IN VIVO.MOLECULAR THERAPY8 3.2003,508-518,具体参见摘要第4-6行,第516页材料与方法的倒数第3、4行. |
KENNETH N, ET AL,.GENIS:GENE EXPRESSION OF SODIUM LODIDESYMPORTER FOR NONINVASIVE IMAGING OF GENETHERAPY VECTORS AND QUANTIFICATION OF GENEEXPRESSION IN VIVO.MOLECULAR THERAPY8 3.2003,508-518,具体参见摘要第4-6行,第516页材料与方法的倒数第3、4行. * |
Also Published As
Publication number | Publication date |
---|---|
US20090285783A1 (en) | 2009-11-19 |
WO2005007109A3 (en) | 2005-07-07 |
JP5550803B2 (ja) | 2014-07-16 |
KR100903729B1 (ko) | 2009-06-19 |
CN1871034A (zh) | 2006-11-29 |
JP2007528715A (ja) | 2007-10-18 |
KR20060054290A (ko) | 2006-05-22 |
WO2005007109A2 (en) | 2005-01-27 |
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